CN110045116A - A kind of viral antigen coating promotor - Google Patents
A kind of viral antigen coating promotor Download PDFInfo
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- CN110045116A CN110045116A CN201811272751.3A CN201811272751A CN110045116A CN 110045116 A CN110045116 A CN 110045116A CN 201811272751 A CN201811272751 A CN 201811272751A CN 110045116 A CN110045116 A CN 110045116A
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- reducing agent
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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Abstract
The invention discloses a kind of viral antigens to be coated with promotor, including reducing agent, protein denaturant, nonionic surfactant and organic reducing agent；Wherein reducing agent includes mercaptoethanol, mercaptopropionic acid or SDS, and protein denaturant includes guanidine hydrochloride or urea, and nonionic surfactant includes Tweem20, and organic reducing agent includes DTT.When practical preparation, the concentration of mercaptoethanol is 2 ‰；The concentration of SDS is 2 ‰；Effect is best when the concentration that the concentration of Tween 20 is 1 ‰, DTT is 1 ‰.Coating promotor prepared by the present invention can greatly improve the coating efficiency of envelope antigen, substantially increase the detectability of positive sample, can be used for the detection to viral disease such as AIDS, hepatitis B etc..
Description
Technical field
The present invention relates to field of virus detection, more particularly, to a kind of viral antigen that can effectively promote antigen coat efficiency
It is coated with promotor.
Background technique
Hepatitis C is one kind as caused by Hepatitis C Virus (HCV) mainly through the disease of blood born, the chronic sense of HCV
Dye can lead to liver chronic inflammation necrosis and fibrosis, some patientss can develop for cirrhosis even hepatocellular carcinoma, 50%-80%'s
Hepatitis C virus infection will progress to chronic states, and wherein the patient of 20%-30% would develop into cirrhosis or liver cancer, to trouble
The health and lives of person generate harm." Chinese viral hepatitis type C nosocomial infection prevention and control guide " is pointed out, hepatitis has high hidden
It hides, the characteristics of height is failed to pinpoint a disease in diagnosis, high chronicity, on the other side to be, China Public is low to the cognitive rate of hepatitis, consultation rate is low, treatment
Rate is low.
The detection means of viral infectious can be carried out from antigen, antibody, nucleic acid etc. are many-sided.Antibody test is current
The important means of hepatitis C primary dcreening operation, but the antigen type that Hepatitis C Virus is generated in patient's body proliferation is more complex, so
It is extremely important to the sensitivity of detection reagent and specificity to search out suitable antigen fragment.
The antigen mainly used at present has Core, NS3, NS4, NS5 etc..Core antigen is the structural proteins of HCV virus,
NS3, NS4, NS5 etc. are the non-structural proteins of HCV virus, and these types of virus protein stimulation body generates the antigenic determinant of antibody
Mostly space structure.It is mostly gene recombinant antigens as albumen used in antibody assay kit, when expression and purification often not
Effective space structure can be formed, keeps coating efficiency extremely low, pattern detection value inaccuracy.
Summary of the invention
The purpose of the present invention is to provide a kind of viral antigens that can improve antigen coat efficiency to be coated with promotor.
To achieve the above object, the present invention can take following technical proposals:
Viral antigen of the present invention is coated with promotor, including reducing agent, protein denaturant, nonionic surfactant and has
Machine reducing agent；Wherein reducing agent includes mercaptoethanol, mercaptopropionic acid or SDS, and protein denaturant includes guanidine hydrochloride or urea, it is non-from
Sub- surfactant includes Tweem20, and organic reducing agent includes DTT.
The reducing agent is mercaptoethanol, and concentration is 0.5 ‰ ~ 5 ‰；The protein denaturant is SDS, and concentration is
0.2‰~5‰；The nonionic surfactant is Tween 20, and concentration is 1 ‰ ~ 1%, and the organic reducing agent is DTT,
Its concentration is 0.5 ‰ ~ 3 ‰.
The concentration of the mercaptoethanol is 2 ‰；The concentration of the SDS is 2 ‰；The concentration of the Tween 20 is 1 ‰, institute
The concentration for stating DTT is 1 ‰.
When being coated with to antigen, mercaptoethanol, SDS and Tween 20 are added in coating buffer first, stirred
After mixing 0.5 hour, envelope antigen is added, continues stirring 0.5 ~ 1 hour, carries out microwell plate coating later, is coated with plate at 2 ~ 8 DEG C
After placing 16 ~ 20 hours, coating plate coating is completed, and is cleaned packet liquid one time using washing lotion, is finally closed, closed with DTT
After 2 ~ 8 DEG C of microwell plate afterwards are placed 16 ~ 20 hours, confining liquid is dried, coating plate is dried.
The anionic surfactant, reducing agent and non-ionic surface active of albuminous degeneration effect is employed herein
Agent may be to influence the principal element of purifying protein space structure variation.
Viral antigen is coated with the promotion of efficiency mainly from the protein steric structural for changing recombinant antigen is adjusted, and is released effectively anti-
Original antibody binding site covers nonessential site, improves the sensitivity of kit clinical detection and specificity, promotes kit detection
Antibody accuracy.
Coating promotor prepared by the present invention can greatly improve the coating efficiency of envelope antigen, substantially increase positive sample
Detectability, can be used for the detection to viral disease such as AIDS, hepatitis B etc..
Specific embodiment
More detailed explanation is done to the present invention below by specific embodiment.Experiment side as used in the following examples
Method is conventional method unless otherwise specified, used in material, reagent etc., unless otherwise specified, commercially
It obtains.
Embodiment 1 prepares a kind of coating promotor that can improve hepatitis C antigen coating efficiency
The coating promotor that the present invention prepares includes mercaptoethanol 2.3ml, 20 1ml and DTT of SDS 2g, Tween
0.3084g。
Hepatitis C antigen method for coating (uses above-mentioned coating promotor):
In use, antigen coat CB buffer is prepared first, by Na2CO31.59g NaHCO3,It is pure that 2.93g is added to 1000ml
Change water constant volume, dissolution sufficiently finishes；Then it is slow mercaptoethanol 2.3ml, 20 1ml of SDS 2g, Tween to be added to coating CB
In fliud flushing, 0.5h is stirred, the amount of required envelope antigen is added in the coating buffer of the promotor containing coating, stirs 0.5h,
Microwell plate coating is carried out according to 100 holes μ l/, coating plate is placed 16 ~ 20 hours at 2 ~ 8 DEG C after coating；
It is coated with plate envelope and protects liquid preparation, by NaH2PO4•2H2O 0.592g, Na2HPO4•12H2O 5.8g, NaCl 9g, trypticase
10g, sucrose 100g are added to 1000ml purified water, and dissolution sufficiently, is protected in envelope and DTT 0.3084g is added in liquid, and dissolution is sufficiently standby
With.
It is coated with plate coating and completes (coating is after 16 ~ 20 hours), packet liquid is cleaned one time using washing lotion, is finally carried out with DTT
Closing dries confining liquid after 2 ~ 8 DEG C of microwell plate after closing are placed 16 ~ 20 hours, and coating plate is dried.
After being coated with the processing of promotor, coating efficiency is obviously improved envelope antigen.
The optimization analysis of 2 present invention coating promotor raw material of embodiment
1, coating sensitivity can be improved in SDS
SDS is that common proteins denaturant explores optimum additive amount in 0.2 ‰ ~ 5 ‰ concentration range.Its experimental result
It see the table below 1.
Table 1
As can be seen from Table 1,2 ‰ SDS concentration can make coating sensitivity higher.
2, mercaptoethanol and Tween 20 can improve sensitivity
In the case where SDS concentration (2 ‰) determine, mercaptoethanol and the optimum additive amount of Tween 20 are explored.It tests knot
Fruit see the table below 2.
Table 2
From the results shown in Table 2, it when mercaptoethanol reaches a certain concentration (such as 0.5 ‰ -5 ‰), adds certain density
Tween 2,0(1 ‰) after, detection sensitivity significantly improves, but excessive Tween 20(is sensitive greater than 1%) can then inhibit to detect
Degree.As it can be seen that 0.5 ‰ -5 ‰ and 1 ‰ -1% be mercaptoethanol and the suitable addition concentration of Tween 20 respectively.
When SDS concentration be 2 ‰, when the concentration that the concentration of mercaptoethanol is 2 ‰, Tween 20 is 1 ‰, detection sensitivity
Highest.
3, the determination of DTT concentration
After the optimum concentration of mercaptoethanol, SDS and Tween 20 determines, the concentration of DTT is further determined that.Test data is such as
Shown in small table 3.
Table 3
In table 3, to DTT setting 0.5 ‰, 1 ‰, 1.5 ‰, 2 ‰, 2.5 ‰, 3 ‰, totally six different concentration carry out experimental judgment,
The results show that the concentration of DTT1 ‰ can effectively keep the stability of coating efficiency and envelope antigen.
Influence of the antigen coat promotor of the present invention of embodiment 3 to coating efficiency
Below using hepatitis C antigen coating mode as subjects, to be control producing tradition coating buffer, to verify this
Application of the antigen coat promotor of invention to being promoted in terms of envelope antigen is coated with efficiency.
Control coating mode and coating mode of the present invention is respectively adopted to detect antibody of HCV positive sample, examines
Surveying result see the table below 4.
Table 4
The results show that being mentioned significantly using the coated hepatitis C antigen coating efficiency of present invention coating promotor is added to
It rises, certain pattern detection values can promote 20 times or more.
Claims (3)
1. a kind of viral antigen is coated with promotor, it is characterised in that: including reducing agent, protein denaturant, non-ionic surface active
Agent and organic reducing agent；Wherein reducing agent includes mercaptoethanol, mercaptopropionic acid or SDS, and protein denaturant includes guanidine hydrochloride or urine
Element, nonionic surfactant include Tweem20, and organic reducing agent includes DTT.
2. viral antigen according to claim 1 is coated with promotor, it is characterised in that: the reducing agent is mercaptoethanol,
Its concentration is 0.5 ‰ ~ 5 ‰；The protein denaturant is SDS, and concentration is 0.2 ‰ ~ 5 ‰；The nonionic surfactant
For Tween 20, concentration is 1 ‰ ~ 1%, and the organic reducing agent is DTT, and concentration is 0.5 ‰ ~ 3 ‰.
3. viral antigen according to claim 2 is coated with promotor, it is characterised in that: the concentration of the mercaptoethanol is
2‰；The concentration of the SDS is 2 ‰；The concentration of the Tween 20 is 1 ‰, and the concentration of the DTT is 1 ‰.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811272751.3A CN110045116A (en) | 2018-10-30 | 2018-10-30 | A kind of viral antigen coating promotor |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811272751.3A CN110045116A (en) | 2018-10-30 | 2018-10-30 | A kind of viral antigen coating promotor |
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| CN110045116A true CN110045116A (en) | 2019-07-23 |
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| CN201811272751.3A Withdrawn CN110045116A (en) | 2018-10-30 | 2018-10-30 | A kind of viral antigen coating promotor |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111579781A (en) * | 2020-05-21 | 2020-08-25 | 深圳市宇诺生物技术有限公司 | Hepatitis C virus antibody detection kit, preparation method and detection method |
| CN111579773A (en) * | 2020-05-29 | 2020-08-25 | 珠海丽珠试剂股份有限公司 | Method for coating magnetic beads with mycoplasma pneumoniae membrane protein antigen |
| CN113009152A (en) * | 2021-02-19 | 2021-06-22 | 山东省大健康精准医疗产业技术研究院 | Glycosylated CD59 enzyme-linked immunoassay kit and preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101336374A (en) * | 2005-11-25 | 2008-12-31 | 比奥-雷德巴斯德有限公司 | Method for identifying the genotype in position 171 of the ovine prion protein as well as kits for implementing said method |
| CN103674657A (en) * | 2013-12-24 | 2014-03-26 | 山东莱博生物科技有限公司 | Kit for processing antigen-antibody immune complex in serum or plasma sample and application of kit |
| CN106093402A (en) * | 2016-05-31 | 2016-11-09 | 湖南康润药业有限公司 | Hepatitis C virus antigen-antibody combined detection kit |
| CN108088727A (en) * | 2016-11-21 | 2018-05-29 | 中国科学院大连化学物理研究所 | The preprocess method of protein in a kind of in vitro body fluid |
-
2018
- 2018-10-30 CN CN201811272751.3A patent/CN110045116A/en not_active Withdrawn
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101336374A (en) * | 2005-11-25 | 2008-12-31 | 比奥-雷德巴斯德有限公司 | Method for identifying the genotype in position 171 of the ovine prion protein as well as kits for implementing said method |
| CN103674657A (en) * | 2013-12-24 | 2014-03-26 | 山东莱博生物科技有限公司 | Kit for processing antigen-antibody immune complex in serum or plasma sample and application of kit |
| CN106093402A (en) * | 2016-05-31 | 2016-11-09 | 湖南康润药业有限公司 | Hepatitis C virus antigen-antibody combined detection kit |
| CN108088727A (en) * | 2016-11-21 | 2018-05-29 | 中国科学院大连化学物理研究所 | The preprocess method of protein in a kind of in vitro body fluid |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111579781A (en) * | 2020-05-21 | 2020-08-25 | 深圳市宇诺生物技术有限公司 | Hepatitis C virus antibody detection kit, preparation method and detection method |
| CN111579773A (en) * | 2020-05-29 | 2020-08-25 | 珠海丽珠试剂股份有限公司 | Method for coating magnetic beads with mycoplasma pneumoniae membrane protein antigen |
| CN113009152A (en) * | 2021-02-19 | 2021-06-22 | 山东省大健康精准医疗产业技术研究院 | Glycosylated CD59 enzyme-linked immunoassay kit and preparation method and application thereof |
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Application publication date: 20190723 |